This invention relates to laser capture microdissection a technique wherein a specimen is visualized under a microscope and then overlaid with a layer of transfer material which when activated by a laser adheres to and extracts out specific targeted elements within the specimen for further processing. More particularly, this disclosure focuses on extracting out samples that are equal in size to or smaller than the activating laser beam. The purpose of this invention is to provide a method and apparatus for reliable microdissection of targets within tissue or other specimen samples, smaller than approximately 10 microns in diameter.
In WO 97/13838 entitled Isolation of Cellular Material Under Microscopic Visualization published Apr. 17, 1997 the statement is made at page 20, line 24 the statement is made:
The size of the tissue transferred, depending upon the needs of the operator, can be varied by changing the diameter of the laser beam and pulse duration. Highly reproducible transfers in the 60 to 700 xcexcm diameter range are easily attainable for procurement of small (100 xcexcm to 1 mm) lesions without the encroachment of adjacent, non-neoplastic cells. In most basic and clinical research studies, procurement of several hundred to several thousand cells is necessary to provide sufficient genetic material for reliable amplification and statistically meaningful analysis. However, since laser beams can be focused to less than one cell diameter, transfers of targeted single cells or even parts thereof is thought possible under the practice of the invention.
In the Application that follows we set forth the solution to the transfer that xe2x80x9cis thought possiblexe2x80x9d mentioned above.
Although the first microdissection patents described a rigid inert substrate to which the thermoplastic polymer was applied which could be used as a pressure plate, the original implementation of LCM employed a freestanding film that was applied to the surface of the tissue by gently pressing the film onto the sample. The film above the tissue section of interest was then heated by a 100-micron diameter beam and melted by pulses from a CO2 laser. The length of the laser pulse (between 100 msec and 630 msec) was chosen so as to allow the irradiated film to come to a steady state temperature rise for sufficiently long times for the polymer to flow into the tissue and form a strong bond by replacing air voids within the desiccated sample. The 630 msec pulses typically used with this system were purposefully chosen to be that long to insure sufficient time after the steady state temperature was reached for the melted polymer (which remains molten until the end of the pulse) to reliably flow into the tissue during the laser pulse. In subsequent work it was shown that equivalent transfers could be achieved with this system and 100 msec pulses, although because of irregular spacing between the polymer surface and the tissue, transfer with 100 msec pulses were less reproducible than with the longer pulses. In practice with this LCM system, the objective was to heat the lower surface of the polymer to a little more than its melting point. The CO2 laser delivered power levels were kept within a factor of two of the threshold power required (range of 25-50 mW delivered to a 100 xcexcm spot on an EVA polymer film 100 xcexcm thick). Thus the tissue captured by the melted polymer was typically exposed to peak temperatures of xcx9c90-100 C for xcx9c500 msec. Using this process damage to DNA, RNA, or proteins in the captured sample was not observed by subsequent molecular analysis.
Short pulses were avoided in LCM so as to insure adequate bond strength. Information from a number of manufacturers of EVA-based thermoplastic adhesives (e.g., hot glue) suggested that using EVA adhesives required maintaining molten joint under pressure for more than one second. In the original CO2 laser LCM designs, the use of a pressure plate (transparent and non absorbing of the laser and visible light) was impractical because of the rarity and expense of materials that transmit CO2 laser wavelengths (9-11 xcexcm). Subsequently the introduction of strongly-absorbing near infrared (xcx9c0.8 xcexcm) dyes soluble in the thermoplastic polymers allowed the transfer film to be focally melted by the pulsed infrared laser diodes (xcx9c0.8 xcexcm) easily focused through transparent substrates to small diameters less than 10 xcexcm in the absorbing thermoplastic film.
Laser capture microdissection occurs where the transfer polymer film is placed on a substrate overlying visualized and selected cellular material from a sample for extraction. The transfer polymer film is focally activated (melted) with a pulse brief enough to allow the melted volume to be confined to that polymer directly irradiated. This invention uses brief pulses to reduce the thermal diffusion into surrounding non-irradiated polymer, preventing it from being heated hot enough to melt while providing sufficient heat by direct absorption in the small focal volume directly irradiated by the focused laser beam. This method can be used both in previously disclosed contact LCM or non-contact LCM, using either condenser-side (or beam passes through polymer before tissue) or epi-irradiation (or laser passes through tissue before polymer). It can be used in configurations in which laser passes through tissue before polymer with and without an additional inert substrate. In its preferred configuration it uses the inertial or elastic confinement of the surrounding un-melted thermoplastic polymer (and the overlying attached substrate) to force expansion of the melted polymer into the underlying tissue target. Utilizing the short pulse protocol, the targeted and extracted material can have a diameter equal to or smaller than the exciting beam even as the optical diffraction limits are approached.
For even greater precision and localization, a series of short xe2x80x9csubthresholdxe2x80x9d pulses can be delivered to the same or immediately adjacent points to just contact specific targets within the laser beam (i.e., a target smaller than the laser beam diameter located in the center of the laser pulse). This utilizes the fact that when a volume of polymer is melted from top to bottom of the absorbing thermoplastic film by a laser pulse, it expands a proportional volume towards the tissue. This volume of polymer expansion can be matched to the volume of the desired target including the initial volume of separation between the polymer and the target either by estimation of average pulse parameters required to accomplish that capture with a single pulse or using a laser pulse roughly half that required for single pulse capture and delivering a series of pulses until a bond with the target is achieved.
The purpose of this invention to provide a method that allows reproducible LCM transfer region of less than 20 microns with greatest precision, maximal efficiency, and minimal duration of thermal transients in the target sample caused by contact with the molten thermoplastic polymer during the laser pulse and subsequently until it cools. We have found that a reliable method for obtaining smaller transfer spot sizes (less Man 10 microns in diameter) involves reducing the pulse width of the laser to less than 1 msec and adjusting the peak power of the laser. These pulse widths and powers minimize damage to the macromolecules in the tissue sample.
We note at the time of filing this application, that the contact and adhering of the activatable material to the sample creates an observable phenomena. The user can actually observe the desired adherence to the sample while adjusting pulse length and power to expand, contract, and even shape the areas of adhesion.